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The relationship between periodontal As increasingly subject to the attention of the medical profession, the existing epidemiological, molecular biology, animal models and in vitro experiments indicate that periodontal disease and As there is a correlation, but periodontal disease occur in the As, mechanism in the development process as well as the role of aspects, especially the significance of human vascular endothelial cell NO signaling pathway is still reported rarely. This experiment maintained by in vitro culture Pg 33277 and HUVEC cell lines, analysis Pg 33277 the HUVEC cell invasion ability, NO generation, eNOS and iNOS protein expression, which explore the the Pg vascular endothelial cell function damage pathway to periodontal provide experimental evidence for the disease in the As in the pathogenesis. The first part of Porphyromonas gingivalis Aeromonas and human vascular endothelial cell lines maintained in vitro cultivation purposes Pg 33277 and HUVEC cell lines in vitro, and the use of certain experimental methods for analysis and identification. Materials and Methods PG 33277 Lyophilized placed in tryptic soy broth culture, the culture process in the anaerobic tank, the use of Gram stain and Columbia blood agar plates detection of Pg purity, using SEM and TEM observation The ultrastructure of Pg fimbriae protein genotype using PCR technology identification Pg. Take HUVEC cell lines thaw recovery and to maintain cultured HUVEC cell morphology was observed under an inverted microscope. Gram stain results Pg oil microscope visible red rod-shaped bacteria, was Gram (-); cultured on Columbia blood agar 5 days after the formation of raised, glossy surface smooth black spherical colonies; SEM visible rod shaped or club-shaped bacteria shape TEM observation Pg visible on the surface of the outer membrane and pilus structure; PCR pilin Genotyping Pg 33277 type I pili protein genotype. Visible under an inverted microscope HUVEC round or polygonal, was a typical arranged like paving stones. The conclusion was confirmed in vitro, we cultured Pg 33277 and HUVEC cell lines, and the growth of a stable, in good condition, to lay the foundation for the next experiment. The experimental research purpose of the second part of Aeromonas Porphyromonas gingivalis invasion of human vascular endothelial cells Pg HUVEC cells were co-cultured After analysis Pg invasion capacity of human vascular endothelial cells. Materials and methods MOI of 1:10, 1:100 Pg 33277 HUVEC cells were co-cultured 4,8,12,24 h, 0.25% trypsin digestion HUVEC, centrifugation, fixed in 2.5% glutaraldehyde solution, 1% osmium tetroxide After fixation, the production of ultra-thin slices, lead citrate and uranyl acetate staining was observed under TEM without Pg infection of HUVEC cells as a negative control group. The results observed in the TEM, Pg able to use its pili adhered to the cell surface of HUVEC invasion to intracellular colonization in cells, Pg no significant pathological changes in the ultrastructure. Conclusion In vitro experiments confirmed Pg capable of adhesion and invasion of vascular endothelial cells to evade the host immune defense response, which may further affect the normal function of vascular endothelial cells play. The third part of Porphyromonas gingivalis Aeromonas Pg HUVEC cells were co-cultured human vascular endothelial cell NO production purposes, analysis of the impact of Pg vascular endothelial cell NO generation. Materials and methods MOI of 1:10, 1:100 Pg 33277 HUVEC cells co-cultured cell supernatant was collected at 4,8,12,24 h, respectively, nitrate reductase method for the determination of cell supernatant NO2- concentration, unaffected the Pg intervention of HUVEC cells as a negative control group, SPSS13.0 software package for each sample data one-way ANOVA. Results within 24 h, Pg MOI of 1:10, 1:100 promote NO production in HUVEC cells, compared with the negative control group, the difference was statistically significant (P lt; 0.05). Conclusion In vitro experiments confirmed, Pg can affect human vascular endothelial cell function in normal play, leading to abnormal endothelial cell NO production, Pg can promote endothelial cell NO generation, which may further lead to vascular endothelial cell dysfunction. The fourth part gums Porphyromonas bacteria the Pg and HUVEC cells were co-cultured human vascular endothelial cells eNOS and iNOS protein expression Objective detection Pg vascular endothelial eNOS and iNOS protein expression. Materials and methods MOI of 1:10, 1:100 Pg 33277 HUVEC cells were co-cultured, RIPA cell lysis buffer on ice total protein extracted HUVEC cells were 4,8,12,24 h after Lorry Protein Assay, respectively. taken 50μg protein samples for SDS-PAGE electrophoresis, electrically transferred After electrophoresis, proteins were transferred to PVDF membranes, were added to rabbit polyclonal anti-human eNOS antibody (1:1000), rabbit anti-human iNOS polyclonal antibody (1: 1000) and mouse anti-human β-Action monoclonal antibody (1:2000) 4 ° C overnight and washed 3 times, TBST plus horseradish peroxidase labeled secondary antibodies room temperature 1.5h, ECL luminescent reagent cartridge color, and finally luminous, developing, exposure, scanning film, JS-300 gel image analyzer target band optical density value SPSS13.0 software package on the relative value of the optical density of each sample, one-way ANOVA, unaffected Pg intervention HUVEC cells as a negative control group. Expression of eNOS and iNOS groups HUVEC cells can be compared with the negative control group, Pg MOI of 1:10, 1:100 intervention HUVEC cells, eNOS in protein expression levels decreased, the level of expression of iNOS protein results WB test results showed that: increase, the differences were statistically significant (P lt; 0.05). Conclusion In vitro experiments confirmed Pg can affect vascular endothelial eNOS and iNOS protein expression levels, on the one hand Pg can inhibit eNOS expression by human vascular endothelial cells, on the other hand, Pg can induce vascular endothelial cell iNOS protein expression. In summary, this study by in vitro MOI of 1:10, 1:100 Pg 33277 HUVEC cells co-cultured, TEM observation Pg invasion of HUVEC cells, nitrate reductase assay Pg NO formation of HUVEC WB detection Pg HUVEC cells eNOS and iNOS protein expression, Pg be able to adhere to the HUVEC cell surface and into the cells, and colonization in the cell's cytoplasm, Pg inhibited HUVEC eNOS protein expression of iNOS protein expression, and ultimately promote the NO secretion of HUVEC cells, thus suggesting that Pg can lead to endothelial dysfunction, which may occur in the As, development play a role in promoting.
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